Maltosyl isothiocyanate (MITC) has been found to be a covalent affinity label for the glucose transporters of the human erythrocyte and the rat adipocyte. Overall objectives of our research are to unequivocally identify the glucose transporters of these cells by purification and reconstitution, to elucidate the mechanisms by which they function, and to determine the mechanism by which insulin enhances glucose transport in insulin-responsive cells. The objective for the coming year are: a. To prepare the glucose transporter of human erythrocytes and rat adipocytes in pure, undegraded form. b. To reconstitute glucose transport with credible activity using purified protein components. c. To begin peptide mapping of the purified transporter and to identify the peptide(s) which are labelled by 14C-MITC in the erythrocyte, the basal adipocyte, and the insulin-stimulated adipocyte. d. To synthesize and test 6'-azido maltose as a photoaffinity label and 6'-isothiocyanato-maltose as a covelant affinity label for the "hypothetical" "in" and "out" conformations of the transporter. e. To examine the source of the enhanced number of MITC-reactive transporters in insulin stimulated fat cells.